Folding Mechanisms of Dihydrofolate Reductase and the Response Regulators

这个项目的目标是对阿尔法/贝塔/阿尔法三明治基序的两个成员的折叠机制进行比较分析，这是生物学中最常见的基序之一。以前NSF支持的关于该基序的一个子类-二氢叶酸还原酶(DHFR)折叠机制的研究表明，这个小的两个结构域的蛋白在四个平行的通道中通过一系列路径上的部分折叠状态进行折叠。相比之下，黄毒素折叠家族的两个成员，即该基序的另一个子类，最初错误折叠为一个旁路中间体，该中间体必须至少部分展开才能进入导致天然构象的生产性过渡态。这种错误折叠反应的分子基础将通过实验和计算方法的结合来探索，这些方法将侧重于确定CHEY、NTRC和Spo0F的这些中间体的结构特征和能量学，这些中间体都是黄毒素折叠的反应调节亚类成员。微秒折叠反应的连续流动(CF)小角X射线散射(SAXS)、时间分辨Forster共振能量转移(TrFRET)和远紫外圆二色谱(CD)测量将提供有关空间性质和全局二级结构的定量信息。脉冲猝灭氢交换方法和质量标记多肽的质谱分析将通过识别定义稳定性核心的片段来增强圆二色数据。突变分析将探索个别侧链在Chey折叠和错误折叠中的作用，特别关注中心β-折叠两侧的一对疏水核心中的大的非极性侧链。链熵在Chey错误折叠反应中的作用将通过创建改变链连接性的排列来测试，同时保持3D结构。对反应调节子折叠机制进行以自然为中心的协作模拟将提供对错误折叠的物种以及它们回溯到自然构象的过程的详细结构洞察。与DHFR中在通路上的亚毫秒级折叠反应的结果进行比较，将为我们提供更多关于与反应调节器的对比的见解。这一多维比较分析的结果有望加强对α/β/α夹心蛋白早期折叠反应的了解，并为黄毒素折叠中的错误折叠反应奠定基础。该项目的更广泛影响具有培训、教育和技术开发的组成部分。本科生、研究生和博士后研究员将接受分子生物物理学方面的培训，包括光谱方法、热力学和动力学、复杂的数据分析、蛋白质工程和蛋白质折叠问题。所采用的几种方法，包括圆二色谱和荧光光谱学，以及开发的数据分析算法，将成为PI系研究生生物物理方法高级专题课程的一部分。在这项研究蛋白质折叠机制的过程中开发的分析工具目前正在世界各地的十几个研究实验室中使用，并将通过生物化学和分子药理学部门赞助的网站更容易获得。在前一个授权期内开发的连续流动混合技术将继续得到优化，并通过合作(用于CF-trFRET和CF-CD)或通过Argonne国家实验室高级光子源的BioCAT光束线(用于CF-SAXS)提供给所有用户。

The goal of this project is to perform a comparative analysis of the folding mechanisms of two members of the alpha/beta/alpha sandwich motif, one of the most common in biology. Previous NSF-supported studies of the mechanism of folding of one sub-class of this motif, dihydrofolate reductase (DHFR), have shown that this small two-domain protein folds via a sequential set of on-pathway partially-folded states in four parallel channels. By contrast, two members of the flavodoxin fold family, another sub-class of this motif, initially misfold to an off-pathway intermediate that must at least partially unfold to access the productive transition state leading to the native conformation. The molecular basis for this misfolding reaction will be probed by a combination of experimental and computational methods that will focus on determining the structural features and energetics of these intermediates for CheY, NtrC and Spo0F, all members of the response regulator sub-class of the flavodoxin fold. Continuous-flow (CF) small-angle x-ray scattering (SAXS), time-resolved Forster resonance energy transfer (trFRET) and far-UV circular dichroism (CD) measurements of microsecond folding reactions will provide quantitative information on dimensional properties and global secondary structure. Pulse-quench hydrogen exchange methodology and mass spectrometric analysis of mass-labeled peptides will enhance the circular dichroism data by identifying the segments that define the cores of stability. Mutational analysis will explore the roles of individual side chains in the folding and misfolding of CheY, with a particular focus on large nonpolar side chains in the pair of hydrophobic cores on either side of the central beta-sheet. The role of chain entropy in the CheY misfolding reaction will be tested by creating permutations that vary the chain connectivity while preserving 3D structure. Collaborative native-centric simulations of response regulator folding mechanisms will provide detailed structural insights into the misfolded species and the process by which they backtrack to the native conformations. Comparisons with the results for the on-pathway sub-millisecond folding reaction in DHFR will provide additional insights into the contrast with response regulators. The results of this multi-dimensional comparative analysis are expected to enhance the understanding of early folding reactions in alpha/beta/alpha sandwich proteins and the basis for the misfolding reactions in the flavodoxin fold.The broader impact of this project has training, educational and technology development components. Undergraduate, graduate and postdoctoral fellows will receive training in molecular biophysics, including spectroscopic methods, thermodynamics and kinetics, sophisticated data analysis, protein engineering, and the protein folding problem. Several of the methods employed, including circular dichroism and fluorescence spectroscopy, and data analysis algorithms developed will form a part of a new advanced topics course in Biophysical Methods for graduate students in the PI's Department. The analytical tools developed during the course of this research on protein folding mechanisms are currently being used in over a dozen research labs around the world, and will be made more accessible via a website sponsored by the Biochemistry and Molecular Pharmacology Department. The continuous-flow mixing technology developed during the previous grant period will continue to be optimized and available to all users by collaboration (for CF-trFRET and CF-CD) or through the BioCAT beamline at the Advanced Photon Source at Argonne National Laboratory (for CF-SAXS).